The present invention relates to liquid chromatographic systems; and more particularly, it relates to solvent mixing in high performance liquid chromatography (HPLC).
Liquid chromatography is an analytical method used to isolate and identify the components of a mixture. Liquid chromatography involves a separation of the components of a mixture in solution by selective adsorption and differences in the rates at which the individual components of the mixture migrate through a stationary medium under the influence of a mobile phase.
In liquid adsorption chromatography, the stationary phase consists of a tubular column packed with an adsorbent material. The mobile phase for carrying an analysis sample through the column, commonly referred to as the "carrier," is a solvent mixture comprising two or more miscible liquids, which is introduced into the column. An equilibrium is established for the individual components of a sample mixture according to the "attraction" of each to the stationary phase and according to the solubility of each in the carrier solvent mixture. The rate at which a solute passes through the column of the chromatograph is dependent upon the equilibria existing, and separations occur where the distributions differ.
All liquid chromatography systems include a moving solvent; a means of producing solvent motion, such as gravity or (in more recently developed equipment) a pump; a means of sample introduction; a fractionating column; and a detector.
Operation of a liquid chromatography system with a carrier of two or more solvents mixed in constant, non-varying proportions is referred to as "isocratic" operation. Sometimes, however, it is desirable to operate the liquid chromatographic system using a carrier in which the ratios of the liquids in the solvent mixture vary over time in accordance with some predetermined gradient. This type of operation is referred to as "gradient elution," and the gradient profiles referred to as solvent programs. Within the category of gradient elution operation, the ratios in the solvent mixture can be made to increase at a fixed rate, i.e., linear gradient, or at an increasing rate of change, i.e., convex gradient, or at a decreasing rate of change, i.e., concave gradient, by appropriate control of the solvent mixing apparatus.
Solvent mixing apparatus to provide the carrier has been accomplished using a multiple, parallel pump arrangement in which each pump meters a different solvent into a common outlet flow line to form a solvent mixture. The total flow rate of the solvent mixture approximately equals the sum of the individual solvent flow rates with the composition of the solvent mixture being determined by the ratios of the flow rates of the individual solvents. Gradient compositions are obtained by control of the pumps to vary the relative flow rates of the pumps during a chromatograph analysis run. Apparatus operating in this manner to form a two-component solvent mixture at a constant rate of flow for introduction into the column of a liquid chromatograph, and providing gradient elution operation, is disclosed in U.S. Pat. No. 3,398,689 issued to R. W. Allington on Aug. 27, 1968. The Model 332 dual-pump gradient liquid chromatograph system of Altex Scientific, Inc., described by M. Savage in "Accuracy and reproducibility in a two-pump gradient HPLC," International Lab., May/June, 1979, pp. 190-199 (1979), also exemplifies metering pump solvent mixing apparatus.
It is conventional in prior art metering pump solvent mixing apparatus to utilize high pressure reciprocating pumps to meter solvents into a common high pressure flow line that feeds solvent mixture directly to the chromatograph column. Such is the teaching of the 3,398,689 patent to Allington. M. Savage diagrams and describes a solvent mixing apparatus having two high pressure pumps metering solvents into a high pressure, dynamic stirring mixing chamber from which the column is directly driven. By "high pressure," reference is thereby being made to a pressure level above ambient or atmospheric pressure which is sufficient to drive a chromatograph column.
At high pressure, accurate solvent mixing is difficult to obtain. For one, reciprocating pumps, which are typically used, produce pressure pulsing upon piston direction reversal when check valve closure occurs. This causes transient flow rate variations that produce mixing errors. Also, accurate speed control of high pressure pumps is difficult to obtain. Particular difficulty is encountered in driving a high pressure pump at low speed, as would be required to obtain a low flow rate in a small percentage solvent mixture. Furthermore, undesired solvent compressibility effects are most pronounced at high pressure. "Compressibility effects" refers to changes in the flow rate of a solvent through a metering pump as back pressure changes during pumping of the solvent. Solvent metering pumps commonly are compensated by a solvent compressibility correction factor based on measured pressure. However, the correction factor does not hold true for all solvents because of varying characteristics (compressibility and viscosity). In HPLC, solvents of widely varying characteristics are encountered. Therefore, as a solvent mixture is being formed, the compressibility effects will produce compositional accuracy errors.
As can readily be appreciated now, use of reciprocating metering pumps at low pressure can enhance accuracy in solvent mixing. The enhancement is most pronounced if solvent mixing is done at essentially ambient or atmospheric pressure.
The use of low pressure metering pumps, however, requires a high pressure pump, referred to as an HPLC pump, in series to drive the chromatograph column. Unless the total flow rate of solvent mixture from the metering pumps is matched with the intake flow rate of the HPLC pump, the composition of the solvent mixture reaching the column may be affected. It is, of course, apparent that if the HPLC pump has a greater flow rate than the total flow rate from the metering pumps, the HPLC pump will develop a vapor lock condition and there will be a total loss of flow to the chromatograph column. But also, if the HPLC pump produces a lesser flow rate than the total flow from the metering pumps, the metering pumps will be operating against the back pressure of the HPLC pump which will affect the individual metering pump flow rates in an unknown manner. Alterations in the metering pump flow rates will, of course, alter the solvent mixture composition, such that the constituents of the solvent mixture introduced to the chromatograph column will be of unknown proportions.
One possible solution to the problem of unequal flow rates would be inclusion of a large volume surge tank. That is, a tank containing enough volume of solvent mixture to accommodate during a chromatograph run surges in either the total flow rate of the metering pumps or in the intake flow rate of the HPLC pump. Although the inclusion of a surge tank would obviate much of the operational difficulty resulting from differences in the flow rates, the surge tank would add substantially to the total volume between the point where the solvents come together and the chromatograph column inlet.
In an effort to obviate the problems attendant high pressure metering pump solvent mixing apparatus, several systems have been developed in which solvent mixing is accomplished by use of a proportioning valve mechanism on the low pressure intake side of a high pressure chromatograph system pump driving the column. Among the first to disclose such solvent mixing apparatus was S. H. Byrne et al, "A Multifunctional Gradient Device for Use in High Speed L.C.," 9 J. Chrom. Sci. 592 (1971). Other disclosures and discussions of such apparatus are found in Modern Practice of Liquid Chromatography, J. Wiley & Sons, Interscience (1976) authored by J. J. Kirkland; Contemporary Liquid Chromatography, J. Wiley & Sons, Interscience (1976) authored by R. P. W. Scott; and 3 Instrumentation for HPLC, pp. 41-62, Elsevier Sci. Pub. Co., Amsterdam (1978) authored by J. F. K. Huber. A single, two-way proportioning valve system is also disclosed in U.S. Pat. No. 4,018,685 issued to Saunders et al on Apr. 19, 1977.
In solvent mixing using a proportioning valve, discrete slugs or pulses of solvent are drawn at low pressure into a mixing chamber. This may be accomplished by gravity flow or by a single low pressure pump. In order to drive the column, a high pressure system pump is required between the mixing chamber and the column. Since a constant flow rate to the column is essential, the system pump typically is one of a design which intakes solvent mixture in gulps and builds a liquid head to discharge. If the intake gulps of the system pump should become synchronized with actuations of the proportioning valve to access one of the solvents, discrete slugs of pure solvent are alternately placed into the flow to the column causing fluctuations in the composition. Because of the lengthy minimum response times for a proportioning valve, cycle time must by necessity be several seconds in duration, so merely speeding up the repetition of valve cycling is not an available solution.
A technique for minimizing the solvent slug mixing problem involving the use of a breather reservoir between the mixer and the chromatograph system pump is embodied in the Tracor Model 980 Liquid Chromatograph. In this technique, solvent mixture is provided to the breather reservoir at a flow rate greater than the intake flow rate of the system pump. The reservoir has an overflow vent to waste. However, because of the expense of some solvents, such technique is not favored in some instances.
Thus, although the advantages of using low pressure solvent mixing in an HPLC are apparent, it has not heretofore been realized in practice with these types of solvent mixing apparatus without substantial sacrifice because of the undesired results of interfacing low pressure solvent mixing apparatus to the required high pressure chromatograph system pump.
As additional information with regard to liquid chromatograph systems having solvent mixing apparatus, it is pointed out that, particularly for systems capable of gradient elution operation, a controller is conventionally included within the system for establishing the composition of the solvent mixture produced and the total flow rate at which it is being provided.
In solvent mixing apparatus comprising solvent metering pumps, each of which provides a flow of one solvent at a rate dependent upon its speed of operation, with the combined flows of the pumps forming a solvent mixture at a total flow rate that is approximately the sum of the individual solvent flow rates, the controller acts to control the speed of the pumps. In the apparatus disclosed in the 3,398,689 patent to Allington, solvent metering pump control is provided by a controller mechanism producing an electrical control signal that is applied to motor control circuits which regulate the power to motors driving the solvent metering pumps. The controller mechanism comprises a potentiometer mechanically operated by linkage connected to a cam cut in a way related to the desired solvent composition with respect to time. Alternatively, accurate speed control of the solvent metering pumps may be provided by a microprocessor-based controller, as exemplified by the Model 332 dual-pump gradient liquid chromatograph system of Altex Scientific, Inc. A dual-pump gradient system using pulse-width modulated signals obtained from a microprocessor to control the operation of stepping motor driven solvent metering pumps is described by M. Savage in "Accuracy and reproducibility in a two-pump gradient HPLC," International Lab., May/June, 1979, pp. 190-199 (1979).
In solvent mixing apparatus comprising a proportioning valve accessing each of a plurality of solvent sources for a predetermined portion of a cycle of operation for time share mixing, with a pump delivering accessed solvents into a mixer from solvent mixture is provided, the rate at which the solvent mixture is provided is dependent upon the speed of the pump. Accordingly, the controller acts to establish solvent mixture composition by controlling the time duration that each solvent is accessed within a cycle of valve operation, and acts to establish the rate at which solvent mixture is being provided by controlling the speed of the solvent delivery pump. Representative controllers for operating proportioning valve-type solvent mixing apparatus are found in U.S. Pat. Nos. 4,063,077 and 4,128,476.